Feedback resistant acetohydroxy acid synthethase mutants

Abstract

The present invention provides nucleotide sequences coding for acetohydroxy acid synthetase (AHAS) mutants, the mutated enzymes themselves and a process for the fermentative production of branched-chain amino acids using these enzymes in specific hosts in which genes which code for the modified acetohydroxy acid synthetase (AHAS) are expressed.

1. An isolated nucleic acid sequence encoding a polypeptide having acetohydroxy acid synthetase (AHAS) activity and encoding a polypeptide that does not contain the sequence Gly Ile Ile at the positions corresponding to residues 20-22 of SEQ ID NO: 2 comprising:
 (a) SEQ ID NO: 1, 
 (b) SEQ ID NO: 3, or 
 (c) a polynucleotide sequence that is at least 95% homologous to SEQ ID NO: 1 or 3; 
 wherein said polynucleotide sequence does not encode a protein comprising the sequence Gly Ile Ile at the positions corresponding to residues 20-22 of SEQ ID NO: 2. 
 
     
     
       2. The isolated nucleic acid sequence of  claim 1 , which comprises (a) SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
       3. The isolated nucleic acid sequence of  claim 1 , which comprises (c) a polynucleotide sequence that is at least 95% homologous to SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
       4. The isolated nucleic acid sequence of  claim 1 , which is at least 98% homologous to SEQ ID NO: 1 or SEQ ID NO: 3. 
     
     
       5. The isolated nucleic acid sequence of  claim 1 , which encodes a polypeptide that is at least 84% homologous with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
       6. The isolated nucleic acid sequence of  claim 1 , which contains a codon encoding Asp and Phe, respectively, in the position corresponding to amino acids 21 and 22 in SEQ ID NO: 2 or SEQ ID NO: 4. 
     
     
       7. A vector comprising the isolated polynucleotide sequence of  claim 1 . 
     
     
       8. The vector of  claim 7  that is Vector pECKA or pECKA/ilvBNC. 
     
     
       9. A host cell comprising the vector of  claim 7 . 
     
     
       10. The host cell of  claim 9  that is  Escherichia coli.    
     
     
       11. The host cell of  claim 9  that is  Bacillus subtilis.    
     
     
       12. The host cell of  claim 9  that is  Corynebacterium  or  Brevibacterium.    
     
     
       13. The host cell of  claim 9 , which is a yeast. 
     
     
       14. The host cell of  claim 9  that is a mammalian or insect cell. 
     
     
       15. The host cell of  claim 9  that has been deposited under accession number DSM15652, DSM15651, or DSM15650. 
     
     
       16. A method for making a polypeptide having acetohydroxy acid synthetase (AHAS) activity comprising culturing or growing the host cell of  claim 9  in a medium suitable for expression of said polynucleotide and recovering a polypeptide having acetohydroxy acid synthetase (AHAS) activity.